MicroRNAs (miRNAs) are an abundant class of short (20-24 nt) endogenous noncoding RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs (for review see for example, Bartel, Cell 2004, 116:281-297). In general, genes encoding miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70 nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.
To date, several hundred miRNAs have been described in humans and a large number of them have been implicated in cancer. miRNAs that have been experimentally shown directly to induce tumor phenotypes (i.e., formation) have been termed “oncomirs”. MiR-21 (also known as MIRN21, miRNA21, hsa-mir-21 and MIR21) is such an oncomir shown to target multiple tumor/metastasis suppressor genes and to have a role in tumor growth, invasion and metastasis. Various studies show that miR-21 is exclusively expressed in cancerous cell lines and solid human tumors, but not in non-transformed cell lines or in the adjacent non cancerous tissue (see for example, Iorio et al., Cancer Res, 2005. 65(16): 7065-7070; Si et al., Oncogene, 2007. 26(19): 2799-2803; Volinia et al., Proc Natl Acad Sci USA, 2006. 103(7): 2257-2261). Thus, evaluation of miR-21 expression has been suggested in cancer diagnosis (see, for example, U.S. Patent Application Publication No. 2006/0105360).
U.S. Patent Application Publication No. 2008/0306018 discloses methods for diagnosing pancreatic cancer comprising measuring the level of at least one miRNA gene product, including miR-21, in a test sample from the subject. This publication further discloses a method of treating pancreatic cancer in a subject in which at least one miRNA gene product is downregulated or upregulated in the cancer cells relative to control cells, inter alia when miR-21 is upregulated in the cancer cells, the method comprising administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miRNA gene product, such that proliferation of cancer cells in the subject is inhibited. Also disclosed are pharmaceutical compositions for treating pancreatic cancer, comprising at least one isolated miRNA gene product or an inhibitor thereof.
Similar methods and compositions have been disclosed for the treatment of lung cancer (U.S. Patent Application Publication No. 2008/0306017), breast cancer (U.S. Patent Application Publication No. 2008/0261908) and solid cancers such as prostate cancer, stomach cancer, pancreatic cancer, lung cancer, breast cancer and colon cancer (U.S. Patent Application Publication No. 2008/0306006). Other exemplary publications which suggest elevating or inhibiting the expression of miR-21 include U.S. Patent Application Publication Nos. 2008/0171715, 2008/0199961 and 2008/0050744. Certain attempts to use miRNAs to control tissue tropism have also been reported (see for example, Barnes et al., Cell Host Microbe, 2008. 4(3):239-248).
Selective targeting of a specific cancer using differentially expressed genes has been previously demonstrated successfully where regulatory sequences of the H19 gene differentially expressed in bladder cancer were used to control the expression of diphtheria toxin (Ohana et al., J Gene Med, 2005. 7(3): 366-374).
WO 99/18195 and U.S. Pat. No. 7,041,654 teach the specific expression of heterologous sequences, particularly genes encoding cytotoxic products (e.g. diphtheria toxin), in tumor cells under the control of a cancer specific promoter (e.g., H19 and IGF promoters).
WO 2007/034487 discloses a nucleic acid construct comprising: (i) a first nucleic acid sequence encoding TNF alpha; (ii) a second nucleic acid sequence encoding a Diphtheria toxin; and (iii) at least one additional nucleic acid sequence comprising a cancer specific promoter (e.g. H19 and IGF promoters); the TNF alpha and Diphtheria toxin encoding sequences being under an expression control of the cancer specific promoter. Also disclosed are construct systems and methods and uses thereof.
WO 2008/087642 discloses compositions and methods for the treatment of cancer and other conditions that are associated with elevated expression of the H19 gene, utilizing constructs encoding H19-silencing nucleic acid agents such as inhibitory RNA.
WO 2006/065938 discloses, inter alia, a method for treating cancer in a mammal comprising administering to the mammal an effective amount of a nucleic acid that encodes an anticancer agent operably linked to a SPANX-N1 promoter.
WO 2003/093441 discloses a method of inhibiting expression of a gene in a cell comprising introducing into said cell a DNA construct comprising a promoter functional in said cell operably linked to a nucleic acid sequence encoding an miRNA precursor having a stem loop structure and comprising in said stem a sequence complementary to a portion of an RNA transcript of said gene, wherein, following introduction of said construct into said cell, said nucleic acid sequence is transcribed and processed so that said miRNA precursor is produced. Further disclosed is a plasmid construct comprising precursor miRNA-21 linked to a CMV promoter.
Cai et al. disclose a plasmid containing the firefly luciferase gene linked to an ˜1 kb DNA fragment of the pri-miR-21 transcription unit corresponding to the region from −959 to +49 relative to the T1 transcription start site (Cai et al., RNA (2004) 10:1957-1966). According to this disclosure, firefly luciferase activity was detected in 293T cells transfected with such a plasmid, leading the authors to conclude that sequences located 5′ to the pri-miR-21 transcription unit can function as an mRNA promoter.
Fujita and Iba disclose a putative promoter region of miR-21 462 nucleotides in length (Fujita and Iba Bioinformatics 2008, 24(3):303-308), and Fujita et al disclose the miR-21 promoter including binding sites for activation protein 1 and PU.1 (Fujita et al., J Mol Biol 2008 May 2; 378(3):492-504).
While several therapeutic approaches utilizing gene therapy in cancer patients have been suggested, there exists a need for additional efficacious anti-cancer agents and vectors. As tumors are known to exhibit significant genomic instability and heterogeneity, the use of hitherto known gene therapy vectors is likely to fail in a substantial number of the patients.
Nowhere in the prior art is it taught or suggested that a miR-21 promoter can be used in recombinant nucleic acid constructs for expressing selectively cytotoxic agents in cancer cells. Nor does the art demonstrate the use of such vectors as an effective and safe anti-cancer treatment in vivo. There remains an unmet medical need for developing gene therapy vectors having enhanced therapeutic activity, minimized toxicity and a broad target range for treating neoplastic disorders.